Fecal DNA testing, based on enzymatic-based analysis of disease-derived DNA in stool, has been emerging as an alternative method for colorectal cancer screening because of its non-invasive, highly sensitive, and patient friendly nature. However, stool posted challenges to enzymatic-based analysis because it contains vast amounts of interfering substances that prevent effective DNA extraction and purification, as well as inhibitors that inhibit enzymatic manipulation of DNA, for example, amplification by polymerase chain reaction (PCR). In other words, stool contains a very large amount of impurities (interfering substances and inhibitors).
Stool comprises debris from foods and numerous metabolites from digestion. While solid matter can be removed with centrifugation, soluble impurities remain in solution containing nucleic acids. Cancer related DNA alterations only comprise a small portion of total stool DNA retrieved. Detecting these rare events requires preparation of highly concentrated DNA so that enough amounts of DNA can be loaded (used) into enzymatic-based analysis assays like PCR.
The methods that are currently used for DNA extraction and purification include alcohol precipitation, binding of nucleic acids to silica in the presence of chaotropic salts, gel filtration, anion exchange, and sequence-specific capture. However, they are not effective in preparing highly concentrated, highly purified DNA from stool, especially from a large stool sample. For example, some inhibitors in stool can be co-isolated along with DNA by these methods because the amount of these inhibitors in stool is so large that the methods for DNA extraction just cannot completely remove them. When DNA is concentrated, these inhibitors are concentrated too, inhibiting enzymatic-based analysis assays and therefore leading to a failure of detecting DNA biomarker from stool. Clearly, to use DNA retrieved from stool as a cancer biomarker, it is important that the amount of inhibitors must be minimized, i.e., they should be present in very low concentrations, which will not inhibit enzymatic-based analysis assays even when highly concentrated DNA prepared from a large stool sample are used for analysis.
One solution to this problem is to remove these impurities as much as possible by other means before or during the process of extracting DNA. Prior art methods include cetyl trimethylammonium bromide (CTAB) treatment, phenol/chloroform extraction, protease treatment, and other inhibitor binding/removing procedures such as the PPVP treatment procedure. However, these prior art methods are either not effective or too complicated to use when DNA is extracted from a large amount of stool.
This current invention is designed to address the issue related to removal of the interfering substances and inhibitors from stool for preparation of highly concentrated, highly purified stool DNA. In one method provided, the binding protein(s) that binds to impurities is added to the stool sample before or during the process of extracting DNA. As a result, these bound impurities are not co-isolated with DNA and will not interfere with DNA extraction as well. In another method provided, the method of using the binding protein(s) to remove impurities is combined with a method using a pre-treatment buffer to further improve the effectiveness of removing the impurities from stool.